Free radicals are generally perceived as highly reactive species that are harmful to the cell. There are, however, a growing number of enzymes known that use carbon-based radicals to catalyze a variety of important metabolic reactions. We are studying two enzymes that use free radicals in quite different ways as model systems to investigate several fundamental aspects of enzyme-mediated radical catalysis, a) How do enzymes generate radicals? b) How is the removal of hydrogen, key step in substrate activation, catalyzed? c) How do enzymes control the rearrangement of reactive substrate-radical intermediates towards productive catalysis rather than harmful side reactions? One important class of radical enzymes uses adenosylcobalamin (coenzyme B12)as a "masked" form of free radical that is liberated by homolysis of the coenzyme cobalt-carbon bond. The radical is used to remove a hydrogen atom from the substrate, thereby activating the substrate towards reaction. We are studying the adenosylcobalamin-dependent isomerization of glutamate to 3-methylaspartate, catalyzed by glutamate mutase, as a paradigm for this class of enzymes. We aim to use mutagenesis to investigate how the protein catalyzes homolysis of the coenzyme and controls radical species at the active site. The kinetic properties of various active site mutants will be examined in detail using a combination of rapid-reaction techniques and the crystal structures of selected mutants will be determined so that changes in structure can be correlated with changes in catalysis. We will also start to investigate the mechanism of the newly discovered glycyl radical enzyme, benzylsuccinate synthase, which is involved in the anaerobic degradation of toluene by various bacteria. The enzyme catalyzes a remarkable reaction - addition of toluene to the double bond of fumarate to form (R)-benzylsuccinate. It is believed to share structural and mechanistic similarities with ribonucleotide reductase and pyruvate formate-lyase, however the chemistry catalyzed is very different. We will examine the kinetic properties of the enzyme and attempt to identify and characterize various radical intermediates involved in the reaction. We will explore the substrate specificity of the enzyme to try and identify mechanism-based inhibitors of the protein and to evaluate the potential of the enzyme to detoxify various aromatic compounds.